PROJECT SUMMARY We recently discovered that the polybasic domain (PDB) of the K-Ras membrane anchor adopts defined dynamic structures on the PM that allow for highly selective interactions with membrane lipids. We also found that PBDs with different primary sequences have the capacity to encode different lipid binding specificities. Thus interactions of PBDs with the plasma membrane (PM) are considerably more complex than simple electrostatics. Further analysis showed that the lipid binding specificity of the K-Ras anchor is a key determinant of signal output. In parallel we made the intriguing discovery that PM potential selectively modulates the diffusional dynamics and spatial organization of specific negatively charged lipids and thereby regulates the extent of K-Ras nanoclustering and signaling. This novel mechanism whereby electrical signals can modulate classical signaling pathways through protein-lipid interactions is the basis of electrical regulation of lipid based signaling platforms. In this new grant we will build on these exciting preliminary observations to further define the molecular details of K-Ras membrane anchor structure and function and study the structural dynamics of other small GTPases on the PM. Our core hypothesis is that the observations we have made with K-Ras are generalizable to other small GTPases that have a PBD-containing membrane anchor. Thus in Aim 1 we will test the hypothesis that the K-Ras PM anchor is substantially more complex than a passive charge detector using a wide-range of experimental techniques and molecular simulations. We will show that PBD sequence and conformational dynamics generate lipid-binding specificity that is critical for function. We will test the generalizability of this hypothesis in Aim 2 by analyzing other members of the Ras superfamily. We will also define the PM spatial organization of the same set of proteins. Aim 3 will investigate the molecular mechanisms and the dependence of membrane interactions of multiple small GTPases on membrane potential. The results will yield new insights into how lipid-binding specificity is encoded in the membrane anchors of small GTPases to render them subject to PM lipid composition and organization. Since small GTPases regulate many aspects of cell biology and their dysfunction is linked to multiple pathologies, including oncogenesis, the results will have wide-ranging implications.